The present invention relates to a polypeptide, more specifically, a polypeptide having an activity of degrading cellulose, which is useful for profitable utilization of biomass. The present invention also relates to a gene that is useful for producing said polypeptide by genetic engineering.
Cellulose is represented by (C6H10O5)n. Materials containing cellulose as the main component are exemplified by woods such as pines, cedars, beeches and poplars; stalks and basts such as hemps, paper bushes, rice straws, bagasse and chaff; seed downs such as cotton; old papers such as newspapers, magazines and corrugated cardboard waste papers; other fibrous wastes; pulps, cellulose powder and the like. Recently, old papers from offices are increasing.
A cellulose molecule has a structure in which D-glucopyranose is connected though xcex2-1,4 bonds and has no side chain. Thus, cellulose is composed of glucose, which can be used as a raw material in alcohol fermentation. If cellulose could be degraded into glucose, it would be possible to produce alcohol, which is useful as a fuel and the like, from old papers or fibrous wastes.
Hydrolysis of cellulose into glucose by an acid method or an enzymatic method has been conducted as a method for degrading (saccharifying) cellulose. In the acid method, cellulose is contacted with hydrochloric acid or sulfuric acid to violently degrade a mass of fibers. Since it is difficult to appropriately determine the hydrolysis conditions, the resulting glucose may further react in the presence of a strong acid. Thus, the acid method has a problem of difficulty in recovering glucose with a high yield and the like. Therefore, the acid method is not utilized practically now. To the contrary, the enzymatic method using a cellulose hydrolase has high reaction selectivity and is advantageous in view of environmental protection. Thus, the enzymatic method has become the mainstream of hydrolysis methods. Various methods have been reported [Wood, B. E., et al., Biothechnology Progress, 13:223-237 (1997); U.S. Pat. No. 5,508,183; Zhao Xin, et al., Enzyme Microbial Technology, 15:62-65 (1993), etc.].
Cellulose hydrolases are exemplified by endoglucanase (EC 3.2.1.4), xcex2-D-glucosidase (EC 3.2.1.21), exo-1,4-xcex2-D-glucosidase (EC 3.2.1.74) and cellobiohydrolase (EC 3.2.1.91). The recommended name of endoglucanase is cellulase, and the systematic name is 1,4-(1,3,1,4)-xcex2-D-glucan 3(4)-glucanohydrolase.
A mixture consisting of endoglucanase, xcex2-D-glucosidase, exo-1,4-xcex2-D-glucosidase, cellobiohydrolase and the like is usually used for hydrolyzing cellulose. Such enzymes cooperatively act on cellulose to degrade it into glucose. Several theories are known for the mode of action. Murao et al. has proposed the following model. First, cleavages are introduced into the non-crystalline regions of cellulose by the action of endoglucanase. Cellobiohydrolase acts on the gaps while destroying the crystal. oligosaccharides are produced by the action of endoglucanase and cellobiohydrolase. The oligosaccharides are degraded by the action of xcex2-D-glucosidase to generate glucose [Sawao Murao et al, xe2x80x9cCellulasexe2x80x9d, pp. 102-104, Kodansha (May 10, 1987)].
In most cases, cellulose does not exist as a single cellulose chain. A structure in which many cellulose chains are assembled through hydrogen bond is formed. In this structure, there are crystalline regions in which a number of cellulose chains are densely clustered and non-crystalline regions in which cellulose chains are sparsely placed. The rate-determining step in a hydrolysis reaction by an enzymatic method is a step of separating and dispersing many cellulose chains in the crystalline regions. Accordingly, conducting a reaction for enzymatic degradation at a high temperature has the following advantages: (1) the reaction proceeds efficiently since cellulose crystals are readily destroyed; (2) risk of contamination with miscellaneous bacteria is little; and (3) cooling prior to an enzymatic reaction is not required in a industrial process which requires heating.
xcex2-D-glucosidase from Pyrococcus furiosus, xcex2-D-glucosidase from Thermococcus sp., endoglucanase and xcex2-D-glucosidase from Thermotoga maritima, endoglucanase and xcex2-D-glucosidase from Thermotoga neapolitana and the like are known as cellulose hydrolases from extreme thermophiles [Bauer, et al., Current Opinion in Biotechnology, 9:141-145 (1998)]. Cloning of xcex2-D-glucosidase gene from an extreme thermophile is described, for example, in U.S. Pat. No. 5,744,345. Cloning of endoglucanase gene from an extreme thermophile is described , for example, in WO 97/44361.
Cellobiohydrolase has been isolated from a thermophilic bacterium, Thermotoga sp. FjSS-B.1 [Ruttersmith, et al., Biochemical Journal, 277:887-890 (1991)]. Since the inhibition constant (Ki) of this enzyme for cellobiose is low (0.2 mM), the enzyme is liable to product inhibition. The specific activity using 4-methylumbelliferyl-xcex2-D-cellobioside as a substrate is 3.6 U/mg. Furthermore, since the bacterium should be cultured at a high temperature under anaerobic conditions, it is difficult to industrially produce the enzyme in large quantities. Additionally, since a gene encoding the enzyme has not been cloned, the enzyme cannot be produced by genetic engineering.
The main object of the present invention is to provide cellobiohydrolase having a high inhibition constant for cellobiose and heat resistance, as well as means to produce said cellobiohydrolase at low cost.
The nucleotide sequence of the entire pyrococcus horikoshii OT3 genomic DNA has been determined [Kawarabayasi, et al., DNA Research, 5:55-76 (1998); Kawarabayasi, et al., DNA Research, 5:147-155 (1998)]. A list of proteins having homologies in amino acid sequences with gene products deduced from the respective open reading frames has been published (http://www.bio.nite.go.jp/ot3db_index.html). Existence of open reading frames encoding polypeptides such as xcex1-amylase, xcex1-mannosidase, xcex2-D-galactosidase, xcex2-D-glucosidase, xcex2-D-mannosidase and endoglucanase in Pyrococcus horikoshii OT3 genome has been predicted based on the comparison of homologies with nucleic acids encoding known cellulose hydrolases in this list. However, existence of an open reading frame having a homology with a nucleic acid encoding a known cellobiohydrolase has not been predicted.
As a result of intensive studies, the present inventors have found that there exists an open reading frame (PH1171) in Pyrococcus horikoshii OT3 genome which encodes a polypeptide having a cellobiohydrolase activity. Unexpectedly, it proved that the polypeptide having the amino acid sequence encoded by the open reading frame has a cellobiohydrolase activity although it has a homology with various endoglucanases including endoglucanase from an archaebacterium AEPIIIa. Furthermore, the present inventors have established a method for producing the polypeptide by genetic engineering. Thus, the present invention has been completed.
The present invention provides the following.
(1) a polypeptide having the amino acid sequence of SEQ ID NO:1 or an amino acid sequence in which one or more amino acid residue is deleted, added, inserted and/or substituted in the amino acid sequence of SEQ ID NO:1 and having a cellobiohydrolase activity;
(2) the polypeptide according to (1) above, which has a thermostable cellobiohydrolase activity;
(3) a nucleic acid encoding the polypeptide according to (1) or (2) above;
(4) the nucleic acid according to (3) above, which has the nucleotide sequence of SEQ ID NO:2;
(5) a nucleic acid encoding a polypeptide having a cellobiohydrolase activity which is capable of hybridizing to the nucleic acid according to (3) above under stringent conditions;
(6) the nucleic acid according to (5) above, which encodes a polypeptide having a thermostable cellobiohydrolase activity;
(7) a recombinant DNA containing the nucleic acid according to any one of (3) to (6) above;
(8) a transformant transformed with the recombinant DNA according to (7) above;
(9) a method for producing the polypeptide according to (1) above, the method comprising culturing the transformant according to (8) above and collecting a polypeptide having a cellobiohydrolase activity from the culture;
(10) a method for degrading a polymer of D-glucopyranose bonded through xcex2-1,4 bonds, the method comprising allowing the polypeptide according to (1) above to act on a polymer of D-glucopyranose bonded through xcex2-1,4 bonds to release cellobiose;
(11) a polypeptide having a cellobiohydrolase activity and having an inhibition constant Ki of 10 mM or more for cellobiose; and
(12) the polypeptide according to (11) above, which retains 20% or more of the cellobiohydrolase activity after treatment at 95xc2x0 C. for 5 hours.